Preliminary Study of in vivo electroporation of VEGF-C Gene Transfer for Lymphedema Model

我们研究了血管内皮生长因子（vegei家族）与癌症特征之间的关系（Mori, Saitou， Kuboi等）。另一方面，接受腋下清扫和乳腺癌术后放疗的患者，其患臂淋巴水肿难以治愈（Kitajima et al.）。我们进行了VEGF-C（淋巴管生长因子）基因转移的实验研究，以确定该疗法对淋巴水肿动物模型的疗效和安全性。方法：采用pCAGGS-VEGF-C表达质粒在体内电穿孔法观察基因转染肌肉的效率。将VEGF-C cDNA插入到含有CAG启动子的pCAGGS表达载体中制备该载体。体内电穿孔法采用基因转移设备CUY21EDIT。用川岛法在大鼠一侧腿部产生手术淋巴水肿。给大鼠注射封闭环状质粒DNA （pCAGGS- vegf - c，对照pCAGGS）各50 μg。进行围度测量，计算差值百分比和围度减小率。取大鼠胫骨前肌标本，10%福尔马林固定于PBS中24h。连续切片进行苏木精-伊红（H-E）和免疫组织化学染色，组织学上评估淋巴管的发现（血管密度和血管面积率）。结果：VEGF组与对照组的百分比差异及围度缩小率。但治疗组与对照组间无显著差异。治疗组淋巴管密度和淋巴管面积率均升高；两组间无显著差异。结论：VEGF-C cDNA在体内电穿孔基因转染可能对淋巴水肿有效。

We have examined the relationship between the vascular endothelial growth factors (VEGE-family) and the character of cancer (Mori, Saitou, Kuboi, et al.). On the other hand, the patients who received axillary dissection and postoperative irradiation for the breast cancer produce the lymphedema of the suffered arm that are difficult to cure (Kitajima et al.). We conducted the experimental study of the gene transfer of VEGF-C (lymphatic duct growth factor) to determine the effect and safety of this therapy for lymphatic edema animal model.Method : The pCAGGS-VEGF-C expression plasmid was used to assess the efficiency of gene transfer into muscle by electroporation in vivo. This vector was prepared by inserting a VEGF-C cDNA into the pCAGGS expression vector containing the CAG promoter. In vivo electroporation method was performed using the gene transfer equipment CUY21EDIT. The surgical lymphedema by the method of Kawashima was created in the leg of one side of the rat. The rat were injected with 50 μg each of closed circular plasmid DNA (pCAGGS-VEGF-C, control pCAGGS). Circumference measurement was performed to calculated percent difference and circumference reduction rate. The tibialis anterior muscles samples for sacrificed rat were fixed 10% formalin in PBS for 24hours. Serial cut sections were sliced for hematoxylin-eosin (H-E) and immunohistochemical staining to evaluate lymphatic vessel findings (vessel density and vessel area rate) histologically. Results : The percent difference and the circumference reduction rate respectively showed between VEGF group and control. However, a significant difference was not found between the treatment group and control group. Lymphatic vessels density and vessel area rate were also increased in treatment group however ; there was no significant difference between the two groups. Conclusion : In vivo electroporation gene transfer of VEGF-C cDNA was demonstrated likelihood of effective to the lymphedema.